Method for preparing microsphere

ABSTRACT

It is to provide an improved method for preparation of microsphere from an emulsion wherein an organic phase containing an organic solvent having a boiling point lower than that of water and a hardly-water-soluble polymer is emulsified in an aqueous phase by an in-water drying method, which comprises: (1) using an apparatus equipped with a gas separation membrane; (2) supplying the emulsion to be subjected to in-water drying to one side of said gas separation membrane; (3) evaporating off the organic solvent contained in said emulsion to the other side of said gas separation membrane, which can remove the organic solvent with high efficiency and can be carried out in a closed system and hence is favorable from the environmental viewpoint.

TECHNICAL FIELD

[0001] The present invention relates to an improved method forpreparation of microsphere by an in-water drying method, moreparticularly, the present invention relates to a method for preparationof microsphere from an emulsion wherein an organic phase containing anorganic solvent having a boiling point lower than that of water and ahardly-water-soluble polymer is emulsified in an aqueous phase by anin-water drying method, which comprises using an apparatus equipped witha gas separation membrane, supplying the emulsion to be subjected toin-water drying to one side of said gas separation membrane, evaporatingoff the organic solvent to the other side of said gas separationmembrane, by which the organic solvent can effectively be removed,particularly said process being able to be carried out in a closedsystem, and hence being useful for preparation of microsphere on anindustrial scale.

BACKGROUND ART

[0002] So-called “an in-water drying method” is known as one of themethods for preparation of microsphere, which comprises dispersing anorganic phase prepared by dissolving a hardly-water-soluble polymer in awater-immiscible organic solvent having a boiling point lower than thatof water in an aqueous phase to prepare an O/W type emulsion, andthereafter removing the organic solvent (cf, for example, JP-B-56-19324;JP-A-63-91325; JP-A-8-151321; Kajeev Jain et al., “Controlled DrugDelivery by Biodegradable Poly (Ester) Devices: Different PreparativeApproaches”, Drug Development and Industrial pharmacy, vol. 24 (No. 8),pp. 703-727, 1998; JP-A-60-100516; JP-A-62-201816; JP-A-9-221417; andJP-A-6-211648).

[0003] This in-water drying method includes a problem of removal of theorganic solvent in view of environmental pollution.

[0004] For example, it is known a method comprising emulsifying adrug-containing solution of a polymer in methylene chloride in anaqueous phase and then removing the organic solvent by stirring theemulsion as it stands at room temperature for a long period of time(cf., JP-A-9-221417, etc.), but according to this method, the methylenechloride is released in the air through the aqueous phase.

[0005] A regulation for prevention of environmental pollution is issuedby Kanagawa Prefecture, whereby the concentration of methylene chlorideat an exhaust port of a factory is regulated less than 50 ppm (cf., “AHandbook for Proper Use of Chlorocarbons” issued by ChlorocarbonSanitary Society, 1996), and further organic solvents such as methylenechloride, chloroform are defined as class I chemical substance whichshall be well controlled by the industrial companies by “A law in regardto monitor of the discharging amount etc. of specific chemicalsubstances into environment and improvement of control thereof”published on Jul. 13, 1999 as well as by an Order of the Governmentissued on Mar. 29, 2000.

[0006] Accordingly, when organic solvents such as methylene chloride areused on an industrial scale, it is required to treat it by using anapparatus in a closed system so that the discharge thereof into outsideshall be well controlled in view of environmental problem.

[0007] Moreover, where the produced microspheres are used as amedicament (particularly, parenteral utilization such as injection,depots), it is essential to sterilize in the step of preparation thereofand it is required to prepare the microspheres in a closed system so asto prevent from contamination of microbes outside.

[0008] For a method for controlling the discharge of organic solvents,it has been studied to prepare microspheres in a closed system. Forexample, a method was proposed where the contact area of an outeraqueous phase and a gaseous phase as well as the rate of circulation andstirring speed of an emulsion are controlled and further a gas is blownonto the emulsion in order to increase the moving speed of gas at thesurface of the emulsion (cf., JP-A-9-221418).

[0009] However, by the method of blowing of gas onto the surface, thesurface area of the emulsion to be contacted with the gas is limited,and hence, it is not enough to remove efficiently the organic solventfor practical use, and further it has very low efficiency of the removalof organic solvent so that a large amount of the aqueous phase isrequired in order to prevent coagulation of the emulsion. Thus, thismethod has a problem that it is difficult to miniaturize the apparatusfor the preparation of microspheres.

[0010] On the other hand, it is also proposed to use a polymer membranefor the purpose of disposal of waste water and recovery of vapor oforganic solvent (cf., Ind. Eng. Chem. Res., vol. 32, p. 533, 1993), andthere have been found some membranes for separation and recovery ofchlorine organic solvents (cf, JP-A-5-15749, JP-A-6-55166,JP-A-7-284641, JP-A-8-57274, JP-A-9-117642).

DISCLOSURE OF INVENTION

[0011] The present invention is to provide an improved method for theindustrial preparation of microspheres by a known in-water dryingmethod, where the removal of the organic solvent, which has been aproblem in the past, can be carried out with high efficiency and furthercan be carried out in a closed system, and hence is favorable from theenvironmental viewpoint.

[0012] The present inventors have found that in a method for preparationof microspheres by an in-water drying method, when an emulsion istreated by using a gas separation membrane and the organic solvent isevaporated off through said gas separation membrane, the desired removalof the organic solvent can effectively be carried out. The presentinvention has been completed based on these new findings.

[0013] That is, the present invention is to provide a method forpreparation of microspheres from an emulsion wherein an organic phasecontaining an organic solvent having a boiling point lower than that ofwater and a hardly-water-soluble polymer is emulsified in an aqueousphase by an in-water drying method, which comprises: (1) using anapparatus equipped with a gas separation membrane; (2) supplying a partor whole of the emulsion to be subjected to in-water drying to one sideof said gas separation membrane; (3) evaporating off the organic solventcontained in said emulsion to the other side of said gas separationmembrane.

[0014] According to the method of the present invention, since themicrospheres can be prepared in a closed system by an in-water dryingmethod, it is advantageously applied to preparation of microspheres, forexample, of medicaments, particularly parenteral preparations such asinjection preparations, depots, which are essentially required to beprepared substantially under an aseptic condition. Besides, since it canbe done in a closed system, there is no problem of releasing of theorganic solvent toward outside, and hence, it is advantageous in thatthere is no problem of environmental pollution.

[0015] Moreover, the removal of the organic solvent can be carried outwith high efficiency in the method of the present invention so that thedesired microspheres can be produced within a very short period of time.It is also advantageous in that undesirable leak of the medicamentcontained in the organic phase into the aqueous phase is effectivelyprevented and further that undesirable coagulation of microsphereshardly occur.

[0016] In addition, according to the method of the present invention,the removal of organic solvent can be done with a small amount ofaqueous phase, and hence, it is possible to miniaturize the apparatus,which can easily be operated in a closed system, and thereby theresistance in stirring of the emulsion, which is a problem forindustrialization of the method, can advantageously be minimized.Moreover, the method of the present invention can continuously proceedby circulating the liquid after evaporating off the organic solvent intothe emulsion, and hence, the method of the present invention isparticularly useful for the preparation of microspheres on an industrialscale.

BRIEF DESCRIPTION OF DRAWINGS

[0017]FIG. 1 is a schematic diagram of a circular type of apparatus forproducing microspheres.

[0018]FIG. 2 is a schematic diagram of an immersing type of apparatusfor producing microspheres.

[0019]FIG. 3 is a schematic diagram of a circular type of apparatus forproducing microspheres provided with emulsifying function.

[0020]FIG. 4 is a schematic diagram of an immersing type of apparatusfor producing microspheres provided with emulsifying function.

[0021]FIG. 5 is a schematic diagram of an immersing type of apparatusfor producing microspheres with a function of controlling temperature.

[0022]FIG. 6 is a graph showing the correlation between the passing rateof nitrogen gas and the rate of removal of methylene chloride in animmersing type of apparatus for producing microspheres.

[0023]FIG. 7 is a graph showing the change with time of the accumulatedamount of taltirelin dissolving out from the microspheres at 37° C.

[0024]FIG. 8 is a graph showing the change with time in the amount ofleuprorelin acetate remained in the microspheres at 37° C.

[0025] [Explanation of Symbols]

[0026] a¹-a⁵: A closed vessel

[0027] b¹, b³: A hollow fiber membrane module

[0028] b², b⁴, b⁵: A cylindrical hollow fiber membrane module

[0029] c¹, c³: A circulating pump

[0030] d¹, d³, d⁵: A stirring blade

[0031] d², d⁴: A magnetic stirring piece

[0032] e¹, e³: A filter

[0033] f¹, f³: A ventilation (suction) hole

[0034] f², f⁴, f⁵: A pathway for passing a gas

[0035] g¹, g³: A circulation pathway

[0036] h², h⁴: A magnetic stirrer

[0037] i³, i⁴: A homogenizer

[0038] j⁵: A jacket for controlling temperature

BEST MODE FOR CARRYING OUT THE INVENTION

[0039] The emulsion to be applied to the method for preparation ofmicrospheres of the present invention is an O/W type emulsion where anorganic phase containing an organic solvent having a boiling point lowerthan that of water and a hardly-water-soluble polymer is emulsified inan aqueous phase, and when the organic phase contains a medicament, animaging material (e.g., pigments, colorants) (cf., JP-A-62-95366,JP-A-62-254833, JP-A-6-118636), there can be obtained microspherescontaining those materials.

[0040] The above “emulsion” can be prepared by various conventionalmethods depending on the kinds of the organic phase, including thefollowing ones.

[0041] (a) An organic phase wherein a medicament, etc. is directlydissolved or dispersed in a solution of a hardly-water soluble polymer,which gives an O/W type emulsion when dispersed into an aqueous phase(cf., JP-B-56-19324, JP-A-63-91325, JP-A-6-32732, JP-A-8-151321, theabove-mentioned literature by Jain et al., etc.)

[0042] (b) An organic phase of a W/O type emulsion wherein an aqueoussolution of a medicament, etc. is dispersed in a solution of ahardly-water soluble polymer, which gives a (W/O)/W type emulsion whendispersed into an aqueous phase (cf., JP-A-60-100516, JP-A-62-201816,JP-A-9-221417, the above-mentioned literature by Jain et al., etc.)

[0043] (c) An organic phase of an O/O type emulsion using two or morekinds of hardly-water-soluble polymers wherein a medicament, etc. isdissolved or dispersed in a solution of one hardly-water solublepolymer, which solution is dispersed in a solution of anotherhardly-water-soluble polymer, which gives an (O/O)/W type emulsion whendispersed into an aqueous phase (cf., JP-A-6-211648)

[0044] The organic solvent having a boiling point lower than that ofwater as used in the above methods includes halogenated aliphatichydrocarbon solvents (e.g., methylene chloride, chloroform, carbontetrachloride, chloroethane, dichloroethane, trichloroethane, etc.),fatty acid ester solvents (e.g., methyl acetate, ethyl acetate, etc.),aromatic hydrocarbon solvents (e.g., benzene), aliphatic hydrocarbonsolvents (e.g., n-hexane, n-pentane, cyclohexane, etc.), ketone solvents(e.g., methyl ethyl ketone, etc.), ether solvents (e.g., diethyl ether,diisopropyl ether, methyl isobutyl ether, methyl tert-butyl ether,tetrahydrofuran, etc.).

[0045] These organic solvents have preferably a boiling point of 15-60°C. lower than that of water under the condition of removal of theorganic solvents, and particularly preferred organic solvents aremethylene chloride, chloroform, and ethyl acetate.

[0046] The hardly-water-soluble polymer used in the present methodincludes various kinds of hardly-water-soluble polymers, but when themicrospheres to be prepared are for the purpose of a medicinal use, itis preferably a biodegradable polymer.

[0047] The biodegradable polymer includes a polyester of a hydroxyfattyacid and derivatives thereof (e.g., polylactic acid, polyglycolic acid,polycitric acid, polymalic acid, poly-β-hydroxybutyric acid,ε-capro-lactone ring opening polymer, lactic acid-glycolic acidcopolymer, 2-hydroxybutyric acid-glycolic acid copolymer, polylacticacid-polyethyleneglycol copolymer, polyglycolic acid-polyethyleneglycolcopolymer, etc.), a polymer of an alkyl α-cyanoacrylate (e.g.,poly(butyl 2-cyanoacrylate), etc.), a polyalkylene oxalate (e.g.,polytrimethylene oxalate, polytetramethylene oxalate, etc.), a polyorthoester, a polycarbonate (e.g., polyethylene carbonate,polyethylenepropylene carbonate, etc.), a polyortho-carbonate, apolyamino acid (e.g., poly-γ-L-alanine, poly-γ-benzyl-L-glutamic acid,poly-γ-methyl-L-glutamic acid, etc.), a hyaluronic acid ester, and thelike, and one or more of these polymers can be used.

[0048] Among these polymers, particularly preferred ones are polyestersof hydroxyfatty acids, which have an average molecular weight of 2,000to about 800,000, more preferably 2,000 to about 200,000.

[0049] Among the above polyesters of hydroxyfatty acids, more preferableones are polylactic acid, lactic acid-glycolic acid copolymer,2-hydroxybutyric acid-glycol acid copolymer. The lactic acid-glycolicacid copolymer has preferably a molar ratio of lactic acid/glycolic acidin the range of 90/10 to 30/70, more preferably 80/20 to 40/60, and the2-hydroxybutyric acid-glycolic acid copolymer has preferably a molarratio of 2-hydroxybutyric acid/glycolic acid in the range of 90/10 to30/70, more preferably 80/20 to 40/60.

[0050] The above hardly-water-soluble polymer is dissolved in an organicsolvent in a concentration in the organic phase of 0.01 to 90% byweight, preferably 0.1 to 70% by weight.

[0051] The O/W type emulsion to be used in the in-water drying method ofthe present invention can be prepared by mixing a solution of the abovehardly-water-soluble polymer in an organic solvent (an organic phase)with water (an aqueous phase) and then emulsifying the mixture. Thepreparation may be carried out by adding the organic phase to theaqueous phase at one time and then emulsifying the resulting mixture, oralternatively by adding the organic phase to a small portion of theaqueous phase, emulsifying the mixture, and further adding the remainingaqueous phase to the resulting emulsion.

[0052] The above emulsifying procedure can easily be carried out by aconventional method, for example, by mixing the organic phase and theaqueous phase with stirring by using a known emulsifying apparatus suchas a propeller stirrer, a turbine impeller mixer, a high-pressureemulsifier, an ultrasonic dispersion mixer, a static mixer, etc.Besides, the emulsification may also be done by other methods such as amembrane emulsifying method, a spraying method.

[0053] The emulsification by a membrane emulsifying method may becarried out by providing a porous membrane (e.g., porous ceramics whichsurface is optionally chemically modified, porous glass, etc.) betweenthe organic phase and the aqueous phase, extruding the organic phaseinto the aqueous phase through fine holes of the porous membrane bygiving a pressure to the polymer solution, if necessary with stirringthe aqueous phase, for example, by a method as disclosed in Journal ofMicroencapsulation, vol. 11 (2), pp. 171-178, 1994.

[0054] Besides, the emulsification by a spraying method may be carriedout by spraying the organic phase onto the aqueous phase with aconventional spraying apparatus. In this method, if necessary, theaqueous phase may be stirred. The spraying apparatus includes, forexample, an air nozzle, a pressure nozzle, an ultrasonic nozzle, arotary atomizer, etc.

[0055] The emulsion thus prepared contains the organic phase and theaqueous phase wherein the aqueous phase is in the ratio of 1 to 10,000parts by volume, preferably 2 to 1,000 parts by volume, per 1 part byvolume of the organic phase.

[0056] In the preparation of the above emulsion, an emulsifier mayoptionally be added to the aqueous phase in order to stabilize theemulsion. The emulsifier includes, for example, an anionic surfactant(e.g., sodium oleate, sodium stearate, sodium laurylsulfate. etc.), anonionic surfactant (e.g., polyoxyethylene sorbitan fatty acid esters(e.g., Tween 80, Tween 60, manufactured by Nikko Chemicals Co., Ltd.),polyethylene castor oil derivatives.(e.g., HCO-60, HCO-50, manufacturedby Nikko Chemicals Co., Ltd.), polyvinylpyrrolidone, polyvinyl alcohol,carboxymethyl cellulose, methyl cellulose, lecithin, gelatin, etc.).

[0057] The emulsifier is added to the above aqueous phase in an amountof 0.001 to 20% by weight, preferably 0.01 to 10% by weight.

[0058] When the organic phase in the emulsion contains a medicament oran imaging material (e.g., pigments, colorants, etc.), etc., there canbe obtained microspheres containing such a medicament, etc. Thesemedicaments etc. may be added in an amount of 0.01 to 60% by weight,preferably 0.1 to 40% by weight, more preferably 1 to 30% by weight,based on the weight of the hardly-water-soluble polymer.

[0059] Besides, for preparing microspheres containing a medicament,etc., those medicament, etc. may previously be pulverized in order toimproving the rate of uptake thereof into the microspheres, which mayvary depending on the kinds of the polymer and the solvent to be used,etc. Moreover, when the medicament, etc. form a salt and hence the rateof uptake thereof into the microspheres is low, they may first beconverted into a free form and then be subjected to the preparation ofmicrospheres.

[0060] The pulverization of the above medicaments, etc. may optionallybe carried out by a conventional pulverization method, that is, mayphysically be pulverized with jet-mill, hammer mill, rotary ball-mill,vibratory ball-mill, beads mill, shaker mill, rod mill, tube mill, etc.,or by wet pulverization under a high pressure, or alternatively, bycrystallization, for example, by dissolving first the medicament, etc.in a solvent, precipitating them by means of regulating the pH, changingthe temperature, components of solvents, etc. and then recovering themby centrifugation or filtration.

[0061] The medicaments to be applied to the present invention includevarious kinds of medicaments, such as antitumor agents, physiologicallyactive peptides, antibiotics, antipyretics, analgesics,antiinflammatories, antitussives, expectorants, sedatives, musclerelaxants, antiepileptics, antiulcers, antidepressants, antiallergicagents, cardiotonics, antiarrythmic agents, vasodilators,antihypertensive diuretics, antidiabetics, antihyperlipidemic agents,anticoagulants, hemostatics, antitubercular agents, hormones,antinarcotic agents, bone resorption inhibitors, promoters ofosteogenesis, antiangiogenetics, antiemetics, vitamins, etc. Specificexamples of these medicaments are listed below.

[0062] Antitumor agents: taxol, bleomycin, methotrexate, actinomycin D,mitomycin C, vinblastine sulfate, vincristine sulfate, daunorubicin,adriamycin, neocercinostatin, cytosine arabinoside, fluorouracil,tetrahydrofuryl-5-fluorouracil, krestin, picibanil, lentinan, tamoxifen,levamisole, bestatin, azimexon, glycyrrhizin, cisplatin, carboplatin,etc.

[0063] Physiologically active peptides: insulin, somatostatin,sandostatin, growth hormone, prolactin, adrenocortical tropic hormone(ACTH), ACTH derivatives, melanocyte stimulating hormone (MSH),thyrotrophin releasing hormone (TRH) and its derivatives (e.g.,taltirelin, etc.), thyroid stimulating hormone (TSH), luteinizinghormone (LH), luteinizing hormone releasing hormone (LHRH) and itsderivatives (e.g., leuprorelin acetate, etc.), follicle stimulatinghormone (FSH), vasopressin, desmopressin, oxytocin, calcitonin,elcatonin, parathyroid hormone (PTH), glucagons, gastrin, secretin,pancreozymin, cholecystokinin, angiotensin, human placental lactogen,human chorionic gonadotropin (HCG), enkephalin, enkephalin derivatives,endorphin, kyotorphin, interferons (e.g., α-, β-, γ-, etc.),interleukins (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.),taftsin, thymopoietin, thymosin, thymostimulin, thymic humoral factor(THF), serum thymic factor (FTS) and its derivatives, and other thymicfactors, tumor necrosis factor (TNF), colony stimulating factors (e.g.,CSF, GCSF, GMCSF, MCSF, etc.), motilin, dinorphin, bombesin,neurotensin, cerulein, bradykinin, urokinase, asparaginase, kallikrein,substance P, insulin-like growth factor (IGF-I, IGF-II), nerve growthfactor (NGF), cell growth factors (e.g., EGF, TGF-α, TGF-β, PDGF, FGFhydrochloride, basic FGF, etc.), bone morphogenetic protein (BMP),neurotrophic factors (e.g., NT-3, NT-4, CNTF, GDNF, BDNF, etc.), bloodcoagulation factors VIII and IX, lysozyme chloride, polymixin B,colistin, gramicidin, bacitracin, erythropoietin (EPO), thrombopoietin(TPO), etc.

[0064] Antibiotics: gentamycin, dibekacin, kanendomycin, lividomycin,tobramycin, amikacin, fradiomycin, sisomicin, tetracyclinehydrochloride, oxytetracycline hydrochloride, rolitetracycline,doxycycline hydrochloride, ampicillin, piperacillin, ticarcillin,cephalothin, cephaloridine, cefotiam, cefsulodin, cefinenoxime,cefinethazole, cefazolin, cefotaxime, cefoperazone, ceftizoxime,moxolactam, thienamycin, sulfazecin, azthreonam, etc.

[0065] Antipyretics, analgesics and anti-inflammatory agents:5-amino-salicylic acid, salicylic acid, sulpyrine, flufenamic acid,diclofenac, indomethacin, morphine, pethidine hydrochloride, levorphanoltartrate, oxymorphone, etc.

[0066] Antitussives and expectorants: ephedrine hydrochloride,methylephedrine hydrochloride, noscapine hydrochloride, codeinephosphate, dihydrocodeine phosphate, alloclamide hydrochloride,chlophedianol hydrochloride, picoperidamine hydrochloride, cloperastine,protokyrol hydrochloride, isoproterenol hydrochloride, salbutamolsulfate, terbutaline sulfate, etc.

[0067] Sedatives: chlorpromazine, prochlorperazine, trifluoperazine,atropine sulfate, methyscopolamine bromide, etc.

[0068] Muscle relaxants: pridinol methanesulfonate, tubocurarinechloride, pancuronium bromide, etc.

[0069] Antiepileptics: phenytoin, ethosuximide, sodium acetazolamide,chlordiazepoxide, etc.

[0070] Antiulcers: metoclopramide, histidine hydrochloride, enprostil,etc.

[0071] Antidepressants: imipramine, clomipramine, noxiptiline,phenelzine sulfate, etc.

[0072] Antiallergic agents: diphenhydramine hydrochloride,chlorpheniramine maleate, tripelenamine hydrochloride, methdilazinehydrochloride, clemizol hydrochloride, diphenylpyraline hydrochloride,methoxyphenamine hydrochloride, etc.

[0073] Cardiotonics: trans-π-oxocamphor, theophylol, aminophylline,etilefrine hydrochloride, denopamine, etc.

[0074] Anti-arrythmia agents: propranolol, alprenolol, bufetorol,oxyprenolol, azimilide, etc.

[0075] Vasodilators: oxyfedrine hydrochloride, diltiazem hydrochloride,tolazoline hydrochloride, hexobendine, bamethan sulfate, etc.

[0076] Antihypertensive diuretics: hexamethonium bromide, pentrilium,mecamylamine hydrochloride, ecarazine hydrochloride, clonidine, etc.

[0077] Antidiabetics: glymidine sodium, glypizide, phenforminhydrochloride, buformin hydrochloride, metformin, etc.

[0078] Anti-hyperlipidemic agents: pravastatin sodium, simvastatin,clinofibrate, clofibrate, simfibrate, bezafibrate, etc.

[0079] Anticoagulants: heparin sodium, etc.

[0080] Hemostatics: thromboplastin, thrombin, menadione sodiumbisulfite, acetomenaphthone, ε-aminocaproic acid, tranexamic acid,carbazochrome sodium sulfonate, adrenochrome monoaminoguanidinemethanesulfonate, etc.

[0081] Anti-tubercular agents: isoniazide, ethambutol, p-aminosalicylicacid, etc.

[0082] Hormones: prednisolone, prednisolone sodium phosphate,dexamethasone sodium hydrochloride, hexestrol phosphate, methimazole,estrone, etc.

[0083] Antinarcotic agents: levallorphan tartrate, nalorphinehydrochloride, naloxone hydrochloride, etc.

[0084] Bone resorption inhibitors: ipriflavone, etc.

[0085] Promoters of osteogenesis: polypeptides such as BMP, PTH, TGF-β,IGF-I, etc.

[0086] Antiangiogenetics: Angiogenesis suppressing steroids, fumagillin,fumagillol derivatives, etc.

[0087] Antiemetics: 5-hydroxytryptamine type 3 receptor antagonists suchas ondansetron or tropisetron, etc., neurokinin 1 receptor antagonists,etc.

[0088] Vitamins: vitamin A, β-carotene, vitamin B₁, vitamin B₂, niacin,nicotinamide, pantothenic acid, calcium pantothenate, vitamin B₆,vitamin B₁₂, folic acid, inositol, para-aminohippuric acid, biotin,vitamin C, vitamin D, vitamin E, vitamin K, etc.

[0089] The medicaments referred to the above may be in free form or bein a pharmaceutically acceptable salt form. For example, when themedicament possesses a basic group such as an amino group, etc., it maybe used in the form of a salt with an inorganic acid (e.g., hydrochloricacid, sulfuric acid, nitric acid, etc.) or with an organic acid (e.g.,carbonic acid, succinic acid, etc.). When the medicament possesses anacidic group such as a carboxyl group, it may be used in the form of asalt with an inorganic base (e.g., alkali metals such as sodium,potassium, etc.) or with an organic base (e.g., organic amines such astriethylamine, basic amino acids such as arginine, etc.).

[0090] The evaporation of the organic solvent through the gas separationmembrane of the present invention is carried out by either one or acombination of two or more of the following methods:

[0091] (1) a method of passing a gas on one side of the gas separationmembrane which is opposite side of supplying the emulsion (“other side”,hereinafter, referred to as merely “the opposite side of emulsion”);

[0092] (2) a method of decompressing said opposite side of emulsion ofthe gas separation membrane; or

[0093] (3) a method of warming the emulsion which is supplied to oneside of the gas separation membrane (hereinafter, referred to as merely“the side of emulsion”).

[0094] In any method, it is preferable to evaporate the organic solventwithout exposing the emulsion to the outside air in view of keeping anaseptic condition and of preventing releasing of the organic solventtoward outside.

[0095] These methods for evaporation of organic solvent are explained inmore detail below.

[0096] (1) A method of passing a gas on the opposite side of emulsion ofthe gas separation membrane:

[0097] The passing of a gas may be carried out by any conventionalmethod such as blowing, suction, etc. and the gas to be passed throughincludes air, nitrogen gas, helium gas, argon gas, etc. Whenmicrospheres for medicinal use are prepared, it is preferable to usesterilized and dust-free (filtered) gas.

[0098] The rate of passing a gas is usually not less than 0.8liter/minute, preferably not less than 6.7 liters/minute, per 1 m² ofthe membrane area.

[0099] (2) A method of decompressing the opposite side of emulsion ofthe gas separation membrane:

[0100] The decompressing is carried out with a decompressing pump, awater-air pump, etc., and the decompressing is carried out so that thedifference of pressure between the side of emulsion and said oppositeside of emulsion is in the range of 1-100 kPa, preferably 20-100 kPa. Itis preferable to control in a manner wherein the side of emulsion isnormal pressure and the opposite side of emulsion becomes reducedpressure.

[0101] (3) A method of warming the side of emulsion:

[0102] The warming is carried out by using a thermostat, an outer bath,a steam bath, etc. so that the emulsion is kept at a prescribedtemperature. The preferred range of temperature may vary depending oncorrelation with a pressure, but it is preferably set at a temperatureof not lower than a boiling point of the organic solvent under thepressure at the opposite side of emulsion, and the temperature isusually in the range of 30 to 80° C., preferably 40 to 60° C., while itmay vary depending on the kinds of the solvent.

[0103] Besides, in any method of the above, in order to removeeffectively the organic solvent from the emulsion, it is preferable tostir the emulsion by a conventional stirring means, such as a screwtype, a magnetic stirrer type, or a paddle type.

[0104] The evaporation of organic solvent by the above methods ispreferably done until the content of the organic solvent in themicrospheres become 35000 ppm or lower. When methylene chloride is usedas an organic solvent, the preparation shall have a final remainingcontent of the solvent of not more than 600 ppm (cf., a Guideline forRemaining Solvent of Medicaments according to International Conferenceon Harmonization of Technical Requirements for Registration ofPharmaceuticals for Human Use among Japan, U.S.A. and Europe (ICH)), butthe organic solvent may further be removed by treatment after producingthe microspheres.

[0105] The gas separation membrane used for the evaporation of the aboveorganic solvent in the method of the present invention includes apervaporation membrane or a porous membrane as mentioned below.

[0106] (1) A Pervaporation Membrane

[0107] It is a non-porous membrane (a homogeneous membrane) which iseffective to separation of a gas taking advantage of the difference inthe dissolution rate of the gaseous molecule in the membrane and therate of diffusion thereof into the membrane. The examples thereof are asilicon-rubber pervaporation membrane (particularly a pervaporationmembrane made of polydimethylsiloxane), a membrane prepared by fillingsilicon rubber into porous polytetrafluoroethylene (cf., JP-A-5-15749),a polyvinyl alcohol mixed membrane (cf., Chemical Engineering, March1998, pp. 25-29). The membranes may be used in a size of 0.1 to 10,000m², preferably 10 to 5,000 m², per 1 m³ of the aqueous phase.

[0108] (2) A Porous Membrane

[0109] This membrane is suitable for separation of the organic solventby utilizing the difference in the flowing rates of the gas moleculeswithin the porous membrane. The examples are a hydrophobic porousmembrane made of polytetrafluoroethylene or polypropylene (cf., Journalof Chemical Engineering of Japan, vol. 31, no. 1, pp. 153-156, 1998), ahollow fiber membrane of ionized crosslinked chitosan/polyacrylonitrilecomposite (cf., Journal of Chemical Engineering of Japan, vol. 25, no.1, pp. 17-21, 1992). The membranes may be used in a size of 0.1 to10,000 m², preferably 10 to 5,000 m², per 1 m³ of the aqueous phase.

[0110] In the method of the present invention, both of theabove-mentioned pervaporation membrane and porous membrane can be used,but the pervaporation membrane is preferable in order to keep an sterilecondition with a closed vessel and to prevent invasion of outsidemicrobes through the membrane.

[0111] Besides, by the treatment of emulsion with these gas separationmembrane, it is preferable to set so that the permeation rate of theorganic solvent through the membrane is larger than the rate of water,but the organic solvent has a boiling point lower than that of water,and hence, when a temperature is appropriately controlled, the organicsolvent can selectively be removed in spite of the permeation rate ofthe gas separation membrane.

[0112] The evaporation of the organic solvent with the above gasseparation membrane can be done by various types of methods as mentionedbelow.

[0113] (1) Circulation Type

[0114] A portion of the emulsion is led to one side of the gasseparation membrane, to which the evaporation treatment of the organicsolvent is applied, and after removal of the organic solvent, theresulting liquid is circulatory returned to the emulsion. In thisprocess, the gas separation membrane is used in the form of a bundle ofplural gas separation membranes which form hollow fibers, in order toenlarge the surface area. It is preferable to introduce the emulsioninto the inner side of the gas separation membrane in the form of hollowfibers.

[0115] Suitable examples of the circular type gas separation membraneare a silicone membrane module (“NAGASEP” manufactured by NagayanagiKogyo Kabushiki Kaisha), a deacrating membrane element (SG-100 series,manufactured by Toray Industries, Inc.), a high performance deaeratingmembrane element (SG-100 series, manufactured by Toray Industries,Inc.), a triple layer composite hollow fibers membrane (a deaeratingmembrane module, manufactured by Mitsubishi Rayon Co., Ltd.), a hollowfiber membrane module (“SEPAREL”, manufactured by Dainippon Ink andChemicals Inc.).

[0116] The circulation of the liquid to be treated is usually carriedout by using a circulation pump in the following manner.

[0117] (a) A method of circulating which comprises taking only a portionof the aqueous phase of the emulsion as a portion of the emulsion

[0118] A portion of the aqueous phase of the emulsion may be carried outby passing the emulsion through a filter (e.g., a stainless mesh filter,a glass filter, a ceramic filter), or by utilizing the difference ofspecific gravity between the organic phase and the aqueous phase byusing an up-and-down liquid-liquid counterflow apparatus (utilizing anextractor manufactured by Tokyo Rikakikai Co., Ltd.). Among thesemethods, a method of passing through a filter is preferably used in caseof less deformability in the organic phase of the emulsion. This methodcomprises circulating a portion of the aqueous phase and hence, it canfavorably be passed through into the inner side of the hollow fiber gasseparation membrane having a smaller diameter of the hollow withoutclogging of the hollow fiber gas separation membrane.

[0119] (b) A method of circulating in the state of an emulsion

[0120] This method comprises circulating a portion of the emulsion aftersubjecting to the evaporation of the organic solvent, where the organicphase of the emulsion contacts the gas separation membrane, and hence,the kinds of the gas separation membrane shall be selected so as to makeit fit to the kind of the organic phase.

[0121] Besides, when a portion of the emulsion is introduced into theinner side of the hollow fiber gas separation membrane, a suitable sizeof the hollow fibers of the hollow fiber gas separation membrane shallbe selected in order to prevent the clogging, and in this case, it ispreferable to decompress the opposite side of emulsion of the gasseparation membrane or to warm the emulsion to be provided into the oneside of the gas separation membrane.

[0122] The method of removal of the organic solvent in the circulationtype can be carried out with an apparatus comprising the following basicelements, and the vessel for filling the emulsion may optionally beequipped with a filter, an up-and-down liquid-liquid counterflowapparatus.

[0123] (i) a vessel for filling an emulsion;

[0124] (ii) a gas separation membrane module for evaporating off anorganic solvent from the emulsion;

[0125] (iii) a circulation pathway which connects the vessel and the gasseparation membrane module; and

[0126] (iv) a pump for circulating the emulsion through the gasseparation membrane module.

[0127] (2) Immersing Type:

[0128] This method comprises immersing an emulsion in a tubular gasseparation membrane, wherein a gas is passed into the inside of thetube.

[0129] It is preferable that the tubular gas separation membrane is inthe form of bundles of plural gas membrane which form hollow fibers andhave a smaller diameter of the tube in order to increase the surfacearea, and that the bundle is immersed in the emulsion and a gas ispassed into the inner side of the tubular gas separation membrane.

[0130] In this method, the organic phase of the emulsion contacts thegas separation membrane, and hence, the kinds of the gas separationmembrane shall be selected so as to make it fit to the kind of theorganic phase.

[0131] The separation membrane is basically the same as the abovecircular type of membrane. However, when the emulsion is stirred forcontacting the emulsion to the gas separation membrane, it is preferableto use a bundle of hollow fiber separation membranes which are figuredin a plate or cylindrical shape.

[0132] The method of removal of the organic solvent with the immersingtype of gas separation membrane is carried out by using an apparatuscomprising basically the following elements:

[0133] (i) a vessel for filling an emulsion; and

[0134] (ii) a gas separation membrane module to be immersed in theemulsion in the vessel for evaporating off an organic solvent from theemulsion.

[0135] (3) Channel Type:

[0136] According to this type of method, the microspheres are preparedby evaporating the organic solvent at the step of passing the emulsionthrough a pathway by

[0137] (a) flowing the emulsion to the outside of the gas separationmembrane where a tubular gas separation membrane is arranged within thepathway and passing a gas through the inner side of the tubular gasseparation membrane in the direction counter to the flow of theemulsion, or

[0138] (b) flowing the emulsion the inner side of the gas separationmembrane where a tubular gas separation membrane is arranged within thepathway and passing a gas through the outside of the gas separationmembrane in the direction counter to the flow of the emulsion.

[0139] According to this method, the microspheres can continuously beproduced without doing each time (batch system) the operations offilling of the emulsion into a vessel, removal of the organic solventand thereafter collecting the microspheres from the vessel in contrastto the circulation type of method or the immersing type of method.

[0140] The apparatus to be used in the process of the present inventioncan be designed appropriately so as to make it fit to the various typesof gas separation membranes but is not limited to any specific one, buta preferred one is illustrated in the working examples hereinafter.

[0141] The organic solvent to be evaporated as mentioned above maypreferably be recovered and re-used. The recovering is done by a methodof liquefying it by cooling, a method of introducing into a cold water,or a method of adsorbing into porous particles. The adsorbing method isdone with an apparatus of adsorbing with fibrous active carbon, ageneral purpose apparatus of recovering chlorocarbon exhaust gas, asmall type of apparatus of recovering chlorocarbon exhaust gas, anapparatus of recovering a low concentration of chlorocarbon exhaust gas,an apparatus of adsorbing with granular active carbon, a fluidized bedapparatus of adsorbing with spherical active carbon, an apparatus ofcompression and condensation by deeply cooling (cf., Handbook for use ofchlorocarbon, pp. 85-93). More specifically, there are used commerciallyavailable apparatuses such as an apparatus of recovering and deodorizingof solvent “Ameig” manufactured by Kurimoto Ltd. and an apparatus foradsorbing and condensing a gas of a solvent in low concentration“Haloneater” manufactured by Toyobo Co., Ltd.

[0142] The microspheres prepared by the process of the present inventionare separated and recovered from the emulsion after evaporation of theorganic solvent by a conventional method such as centrifugation,filtering, or screening.

[0143] Moreover, when the microspheres are heated over the boiling pointof the organic solvent in an aqueous phase (cf., JP Application No.11-39599) or dried with heating after covering with an additive having ahigh melting point (cf., JP-A-9-221417), the remaining organic solventcan be removed.

[0144] The microspheres thus obtained are further washed to remove theadditives of the aqueous phase, etc. which are adhered on the surface,and adding an agglomeration preventing agent such as saccharides, sugaralcohols, inorganic bases, preferably mannitol, sorbitol, in order toprevent the agglomeration among the microspheres, and then subjected tolyophilization.

[0145] Moreover, in order to obtain microspheres having the desiredparticle size, the product is preferably subjected to screening, forexample a screen of 1,000 μm or lower. Particularly, the microspheresare used as an injection preparation, the microspheres are preferablypassed through a screen of 150 μm or lower in order to easily passthrough the needle.

[0146] The microspheres prepared by the method of the present inventionmay be incorporated with medicaments, imaging materials (e.g., pigment,colorant) by adding these medicaments, imaging materials into theorganic phas during the preparation of the microspheres, and hence, canbe used for medicaments, non-carbon papers, aqueous ink, or the like byselecting the additives.

[0147] In case of microspheres containing medicaments, they may be inthe form of fine granules, suspensions, embedded type preparations,injections, preparations applying to the skin, and can be administeredorally or parenterally [intramuscular injection, subcutaneous injection,administration into blood vessel, percutaneous administration,administration via viscous membrane (per oral cavity, per vaginaladministration, per rectal administration, etc.)].

[0148] When the medicament-containing microspheres are used as aninjection preparation or a suspension preparation (e.g., dry syrup fororal administration), they may preferably be prepared in the form of aliquid preparation by incorporating a dispersing agent (e.g., nonionicsurfactants, polyethylene castor oil derivatives, cellulose thickeners),or alternatively, they may be dispersed in water by adding thedispersing agent as mentioned above and an excipient (e.g., mannitol,sorbitol, lactose, glucose, xylitol, maltose, galactose, sucrose), andsolidified by lyophilization, drying under reduced pressure, spraydrying, etc., and the solidified preparation is dissolved in distilledwater for injection when used.

[0149] The above injection preparation may further optionally beincorporated by preservatives (e.g., methylparaben, propylparaben,benzyl alcohol, chlorobutanol, sorbic acid, boric acid, etc.), isotonicagents (e.g., sodium chloride, glycerin, sorbitol, glucose, etc.), pHadjustors (e.g., sodium hydroxide, potassium hydroxide, hydrochloricacid, phosphoric acid, citric acid, oxalic acid, carbonic acid, aceticacid, arginine, lysine, etc.).

EXAMPLES

[0150] The present invention is illustrated by the following Examples,Comparative Examples, Experiments, but should not be construed to belimited thereto.

[0151] (Example of Apparatuses)

[0152] The apparatuses, which may be used for preparing microspheres ofthe present invention are illustrated in FIG. 1 to FIG. 5.

[0153] In order to prepare the microspheres by using the apparatus asshown in FIG. 1, the emulsion to be subjected to an in-water drying isfilled in a closed vessel (a¹), and a portion of the aqueous phase ofthe emulsion passed through a filter (e¹) (e.g., a stainless mesh)provided at the middle of the closed vessel (a¹) is circulated withstirring with a stirring blade (d¹) by passing through a hollow fibermembrane module by the use of a circulation pump (c¹) via a circulationpathway (g¹) made of a silicone rubber, etc. During the circulation,when the portion of the aqueous phase is passed through the hollow fibermembrane module, the organic solvent contained therein is passed andpermeated through the hollow fiber membrane and is evaporated throughthe ventilation (or suction) hole (f¹) and thereby the organic solventcan be efficiently evaporated off to out of the system, and thereby themicrospheres are formed above the filter (e¹) provided in the closedvessel (a¹).

[0154] For preparing microspheres by using an apparatus as shown in FIG.2, the emulsion to be subjected to an in-water drying is filled in aclosed vessel (a²), and a cylindrical hollow fiber membrane module (b²)is immersed in the emulsion. In this situation, air is passed via aventilation pathway (f²) with stirring the emulsion by a magneticstirrer (h²) and a magnetic stirring piece (d²), whereby the organicsolvent in the emulsion is passed and permeated through the hollow fibermembrane and is efficiently evaporated off to out of the system, andthereby the microspheres are formed in the closed vessel (a²).

[0155] For preparing microspheres by using an apparatus as shown in FIG.3, the oil phase and aqueous phase of the emulsion to be subjected to anin-water drying are filled in a closed vessel (a³) and emulsified with ahomogenizer (i³). After emulsifying, a portion of the aqueous phase ofthe emulsion passed through a filter (e³) (e.g., a stainless mesh)provided at the middle of the closed vessel (a³) is circulated withstirring with a stirring blade (d³) by passing through a hollow fibermembrane module by the use of a circulation pump (c³) via a circulationpathway (g³) made of a silicone rubber, etc. During the circulation,when the portion of the aqueous phase is passed through the hollow fibermembrane module, the organic solvent contained therein is passed andpermeated through the hollow fiber membrane and is evaporated throughthe ventilation (or suction) hole (f³) and thereby the organic solventcan be efficiently evaporated off to out of the system, and thereby themicrospheres are formed above the filter (e³) provided in the closedvessel (a³).

[0156] For preparing microspheres by using an apparatus as shown in FIG.4, the oil phase and aqueous phase of the emulsion to be subjected to anin-water drying are filled in a closed vessel (a⁴) and emulsified with ahomogenizer (i⁴). After emulsifying, a cylindrical hollow fiber membranemodule (b⁴) is immersed in the emulsion. In this situation, air ispassed via a ventilation pathway (f⁴) with stirring the emulsion by amagnetic stirrer (h⁴) and a magnetic stirring piece (d⁴), whereby theorganic solvent in the emulsion is passed and permeated the hollow fibermembrane and is efficiently evaporated off to out of the system, andthereby the microspheres are formed in the closed vessel (a⁴).

[0157] For preparing microspheres by using an apparatus as shown in FIG.5, the emulsion to be subjected to an in-water drying is filled in aclosed vessel (a⁵) and a cylindrical hollow fiber membrane module (b⁵)is immersed in the emulsion. In this situation, air is passed via aventilation pathway (f⁵) with stirring the emulsion by a stirring blade(d⁵), whereby the organic solvent in the emulsion is passed andpermeated the hollow fiber membrane and is efficiently evaporated off toout of the system, and thereby the microspheres are formed in the closedvessel (a⁵). In this apparatus, the temperature of the emulsion can becontrolled by a temperature controlling jacket (j⁵), and hence, theorganic solvent can more effectively be evaporated off to out of thesystem.

[0158] In the apparatuses of FIG. 1 to FIG. 5, the ventilation (suction)hole (f¹), (f³) and the ventilation pathway (f²), (f⁴), (f⁵) mayoptionally be connected to an apparatus for recovering the organicsolvent which has functions of cooling, adsorbing, etc.

Example 1

[0159] (1) To a lactic acid-glycolic acid copolymer (average molecularweight: 10,000; lactic acid : glycolic acid=1:1; PLGA 5010, manufacturedby Wako Pure Chemical Industries, Ltd.) (0.45 g) is added methylenechloride (specific grade reagent, manufactured by Katayama ChemicalIndustries, Ltd.) (0.75 g), and the mixture is mixed well with a mixer(Touch Mixer MT-51, manufactured by Yamato) to give a homogeneoussolution, which is used as an oil phase.

[0160] The oil phase is added to a 0.5% aqueous solution of polyvinylalcohol (Poval 220C, manufactured by Kuraray Co., Ltd.) (3 mL), and themixture is emulsified with a homogenizer (POLYTRON Homomixer; KinematicaAG, Littau, the diameter of tip: 7 mm) at 2,500 rpm for 5 minutes togive an O/W type emulsion.

[0161] (2) The emulsion is poured into a cylindrical closed vessel(inner diameter: 80 mm, inner volume: 800 mL) of the apparatus as shownin FIG. 1 wherein 500 mL of water is previously added, and it is stirredwith four stirring blades (diameter: 50 mm, propeller R type,manufactured by HEIDON) which are provided onto a three-one motor(BL-600, manufactured by HEIDON) at 400 rpm at room temperature for onehour.

[0162] Thereafter, simultaneously with stirring with four stirringblades, the aqueous phase passed through a stainless mesh filter(opening size: 20 μm) provided at a lower part of the vessel isintroduced into a silicone rubber hollow fiber membrane module, wherebymethylene chloride is evaporated off under reduced pressure (outerpressure: 80 kPa) to the outside of the hollow fiber membrane, and theaqueous phase passed through the silicone rubber hollow fiber membranemodule is returned to the vessel. The circulation is continued at a rateof 250 mL/minute for 2 hours.

[0163] The used silicone rubber hollow fiber membrane module is NAGASEPM40-A (manufactured by Nagayanagi Kogyo Kabushiki Kaisha) of thefollowing specifications: Thickness of the hollow fiber membrane: 40 μmInner diameter of the hollow fiber membrane: 170 μm Number of hollowfibers: 3000 Effective area of the hollow fiber membranes: 0.3 m²

[0164] (3) The contents of the closed vessel are passed through a meshdefined by Japanese Pharmacopeia (100 mesh, opening size: 150 μm) andthereafter the microsphere particles are separated with a stainless meshfilter (opening size: 20 μm). The microsphere particles thus obtainedare washed with distilled water several times and then transferred to aglass-made sample tube (the full volume: 5 mL, manufactured by IuchiSeieido) and thereto is added a small amount of distilled water, and themixture is lyophilized with a lyophilizer (RLE-52ES, manufactured byKyowa Shinku Co.) at −20° C. for 3 hours (outer pressure: not more than0.3 kPa) and at 20° C. (outer pressure: not more than 0.3 kPa) for 15hours to give microsphere particles (average particle size: 60 μm).

[0165] The microsphere powder thus obtained (0.01 g) is dissolved inchloroform (for high performance liquid chromatography, manufactured byKanto Kagaku K. K.) (10 mL) to give a test sample liquid. The testsample liquid (2 μL) is measured with a gas chromatogram apparatus (themain body GC-14B, Integrator CR-7A, manufactured by ShimadzuCorporation) [column packing; Gaschropack 54 (manufactured by GLScience), column temperature: 150° C.; the detector: FID; injectiontemperature: 170° C.; mobile gas: nitrogen gas; flow rate: 60 mL/h], andbased on a calibration curve previously prepared with a standardsolution of methylene chloride in chloroform, the concentration of thetest sample liquid is estimated, and then in the light of the weight ofmicrosphere particles to be used, the content of the methylene chloridein the microsphere particles are calculated. As a result, it was 25,000ppm.

Example 2

[0166] In the same manner as described in Example 1 except that thestirring period of time is for 30 minutes and the circulation period oftime in the silicone rubber hollow fiber membrane module is for onehour, microsphere particles having an average particle size of 69 μm areproduced.

[0167] Besides, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example1-(3). As a result, it was 30,000 ppm.

Example 3

[0168] (1) The O/W type emulsion obtained in Example 1-(1) is pouredinto a cylindrical closed vessel (inner diameter: 80 mm, inner volume:800 mL) of the apparatus shown in FIG. 1 wherein 500 mL of water ispreviously added, and it is stirred with four stirring blades (diameter:50 mm, propeller R type, manufactured by HEIDON) which are provided ontoa three-one motor (BL-600, manufactured by HEIDON) at 400 rpm at roomtemperature, and simultaneously, the aqueous phase passed through astainless mesh filter (opening size: 20 μm) provided at a lower part ofthe vessel is introduced into a silicone rubber hollow fiber membranemodule, whereby methylene chloride is evaporated off under reducedpressure (outer pressure: 80 kPa) to the outside of the hollow fibermembrane, and the aqueous phase passed through the silicone rubberhollow fiber membrane module is returned to the vessel. The circulationis continued at a rate of 250 mL/minute for one hour.

[0169] The used silicone rubber hollow fiber membrane module is the sameas used in Example 1-(2).

[0170] (2) In the same manner as described in Example 1-(3), thecontents of the closed vessel are treated to give microsphere particleshaving an average particle size of 66 μm.

[0171] Besides, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example1-(3). As a result, it was 32,000 ppm.

Example 4

[0172] (1) The O/W type emulsion obtained in Example 1-(1) is pouredinto a cylindrical closed vessel (inner diameter: 80 mm, inner volume:800 mL) of the apparatus shown in FIG. 1 wherein 500 mL of water ispreviously added, and it is stirred with four stirring blades (diameter:5 cm, propeller R type, manufactured by HEIDON) which are provided ontoa three-one motor (BL-600, manufactured by HEIDON) at 200 rpm at roomtemperature, and simultaneously, the aqueous phase passed through astainless mesh filter (opening size: 20 μm) provided at a lower part ofthe vessel is introduced into a silicone rubber hollow fiber membranemodule, whereby methylene chloride is evaporated off under reducedpressure (outer pressure: 80 kPa) to the outside of the hollow fibermembrane, and the aqueous phase passed through the silicone rubberhollow fiber membrane module is returned to the vessel. The circulationis continued at a rate of 100 mL/minute for one hour.

[0173] The used silicone rubber hollow fiber membrane module is the sameone as used in Example 1-(2).

[0174] (2) In the same manner as described in Example 1-(3), thecontents of the closed vessel are treated to give microsphere particleshaving an average particle size of 64 μm.

[0175] Besides, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example1-(3). As a result, it was 30,000 ppm.

Example 5

[0176] (1) To a mixture of fine particles (0.2 g) of vitamin B₁₂(average diameter: 3 μm, manufactured by Rhône-Poulenc) and a lacticacid-glycolic acid copolymer (average molecular weight: 10,000; lacticacid: glycolic acid=1:1; PLGA 5010, manufactured by Wako Pure ChemicalIndustries, Ltd.) (1.8 g) is added methylene chloride (3 g), and themixture is mixed well with a bath type sonicator (Sonorex Super RK514BH,manufactured by Banderine) to give a homogeneous dispersion, which isused as an oil phase.

[0177] The oil phase is added to a 0.5% aqueous solution of polyvinylalcohol (Poval 220C, manufactured by Kuraray Co., Ltd.) (8 mL), and themixture is emulsified with a homogenizer (POLYTRON Homomixer; KinematicaAG, Littau, the diameter of tip: 10 mm) at 2,500 rpm for 5 minutes togive an O/W type emulsion.

[0178] (2) The emulsion is poured into a closed vessel (inner diameter:80 mm, inner volume: 800 mL) of the apparatus shown in FIG. 1 wherein500 mL of water is previously added, and it is stirred with fourstirring blades (diameter: 50 mm, propeller R type, manufactured byHEIDON) which are provided onto a three-one motor (BL-600, manufacturedby HEIDON) at 400 rpm at room temperature for 30 minutes.

[0179] Thereafter, simultaneously with stirring with four stirringblades, the aqueous phase passed through a stainless mesh filter(opening size: 20 μm) provided at a lower part of the vessel isintroduced into a silicone rubber hollow fiber membrane module asdescribed in Example 1, whereby methylene chloride is evaporated offunder reduced pressure (outer pressure: 80 kPa) to the outside of thehollow fiber membrane, and the aqueous phase passed through the siliconerubber hollow fiber membrane module is returned to the vessel. Thecirculation is continued at a rate of 250 mL/minute for one hour.

[0180] (3) The contents of the closed vessel are treated in the samemanner as described in Example 1-(3), red color microsphere particles(average particle size: 70 μm) are obtained.

[0181] The microsphere particles thus obtained (0.01 g) are weighed andthereto is added acetonitrile (5 mL) and the microsphere particles aredissolved. To the solution is added a 0.5 M aqueous sodium chloridesolution (10 mL), and the mixture is subjected to centrifugation at2,000 rpm for 5 minutes to separate the precipitates.

[0182] As to the resulting supernatant, an absorbance at 360 nm ismeasured with a spectrophotometer (UV-2500PC, manufactured by ShimadzuCorporation), and based on a calibration curve previously prepared, thecontent of vitamin B₁₂ was estimated, and then in the light of theweight of microsphere particles, the content of vitamin B₁₂ in themicrosphere particles was calculated. As a result, it was 7.8%.

[0183] Besides, when it was calculated based on the amount of vitaminB₁₂ used, the content of vitamin B₁₂ in the microsphere particles andthe produced amount of microsphere particles, 78% of vitamin B₁₂ usedwas taken into the microsphere particles.

[0184] Moreover, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example1-(3). As a result, it was 21,000 ppm.

Example 6

[0185] (1) To a mixture of fine particles (0.25 g) of vitamin B₁₂(average diameter: 3 μm, manufactured by Rhône-Poulenc) and a lacticacid-glycolic acid copolymer (average molecular weight: 10,000; lacticacid: glycolic acid=1:1; PLGA 5010, manufactured by Wako Pure ChemicalIndustries, Ltd.) (9.75 g) is added methylene chloride (specific gradereagent, manufactured by Katayama Chemical Industries, Ltd.) (15 g), andthe mixture is mixed well with a bath type sonicator (Sonorex SuperRK514BH, manufactured by Banderine) to give a homogeneous dispersion,which is used as an oil phase.

[0186] The oil phase is added to a 0.5% aqueous solution of polyvinylalcohol (Poval 220C, manufactured by Kuraray Co., Ltd.) (40 mL), and themixture is emulsified with a homogenizer (POLYTRON Homomixer; KinematicaAG, Littau, the diameter of tip: 20 mm) at 2,500 rpm for 5 minutes togive an O/W type emulsion.

[0187] (2) The emulsion is poured into a cylindrical closed vessel(inner diameter: 110 mm, inner volume: 1 L) of the apparatus shown inFIG. 2 wherein 500 mL of water is previously added, and it is stirredwith a magnet stirrer (IS-3DS, manufactured by Ikeda Rika) and aTefron-coated stirring piece (40 mm in size) at 600 rpm at roomtemperature, and simultaneously, methylene chloride is evaporated offfrom the vessel by using a cylindrical silicone rubber hollow fibermembrane module inserted within the vessel, wherein nitrogen gas ispassed through the inside of the hollow fibers. The nitrogen gas ispassed at a rate of 4.5 L/minute. This treatment is carried out for onehour.

[0188] The used cylindrical silicone rubber hollow fiber membrane moduleis NAGASEP M60-1800 in a cylindrical shape of the followingspecifications: Diameter of the cylinder: 100 mm Dimension of thecylinder: 120 mm × 120 mm Thickness of the hollow fiber membrane: 60 μmInner diameter of the hollow fiber membrane: 200 μm Outer diameter ofthe hollow fiber membrane: 320 μm Number of hollow fibers: 1800Effective area of the hollow fiber membranes: 0.15 m²

[0189] (3) The contents of the closed vessel are treated in the samemanner as described in Example 1-(3) to give red color microsphereparticles (average particle size: 59.6 μm) (yield: 78%).

[0190] By calculating in the same manner as described in Example 1-(3)and Example 5-(3), the content of vitamin B₁₂ and the content ofmethylene chloride in the microsphere particles were 2.3% and 36,000ppm, respectively, and 90.3% of vitamin B₁₂ used was taken into themicrosphere particles.

Example 7

[0191] (1) To a mixture of fine particles (0.1 g) of vitamin B₁₂(average diameter: 3 μm, manufactured by Rhône-Poulenc) and a lacticacid-glycolic acid copolymer (average molecular weight: 10,000; lacticacid glycolic acid=1:1; PLGA 5010, manufactured by Wako Pure ChemicalIndustries, Ltd.) (0.9 g) is added methylene chloride (2 g), and themixture is mixed well with a bath type sonicator (Sonorex Super RK514BH,manufactured by Banderine) to give a homogeneous dispersion, which isused as an oil phase.

[0192] The oil phase is entered into a cylindrical closed vessel (innerdiameter: 80 mm, inner volume: 800 mL) as shown in FIG. 3, wherein a0.5% aqueous solution of polyvinyl alcohol (Gosenol EG-40, manufacturedby The Nippon Synthetic Chemical Industry Co., Ltd.) (400 mL) ispreviously filled, and it is emulsified with a homogenizer (POLYTRONHomomixer; Kinematica AG, Littau, the diameter of tip: 20 mm) at 8,000rpm for 3 minutes to give an O/W type emulsion.

[0193] (2) The emulsion is stirred with four stirring blades (diameter:50 mm, propeller R type, manufactured by HEIDON) which are provided ontoa three-one motor (BL-600, manufactured by HEIDON) at 400 rpm at roomtemperature for 30 minutes.

[0194] Thereafter, simultaneously with stirring with four stirringblades, the aqueous phase passed through a stainless mesh filter(opening size: 20 μm) provided at a lower part of the vessel isintroduced into a silicone rubber hollow fiber membrane module asdescribed in Example 1, whereby methylene chloride is evaporated offunder reduced pressure (outer pressure: 80 kPa) to the outside of thehollow fibers, and the aqueous phase passed through the silicone rubberhollow fiber membrane module is returned to the vessel. The circulationis continued at a rate of 250 mL/minute for one hour.

[0195] (3) The contents of the closed vessel are transferred into a 50mL volume Teflon-made centrifuge tube and centrifuged with a centrifugalseparator (KN-30F, manufactured by Kubota Shoji K.K.) at 2,000 rpm for 5minutes, and the supernatant is discharged. The precipitated particlesare washed with a small amount of water, and subjected to thecentrifugation like the above. This centrifugation is repeated threetimes, and the finally obtained microsphere particles are lyophilized inthe same manner as described in Example 1 to give red color microsphereparticles (average particle size: 34.8 μm).

[0196] By calculating in the same manner as described in Example 1-(3)and Example 5-(3), the content of vitamin B₁₂ and the content ofmethylene chloride in the microsphere particles were 7.9% and 13,500ppm, respectively, and 79.0% of vitamin B₁₂ used was taken into themicrosphere particles.

Example 8

[0197] (1) To a mixture of fine particles (0.1 g) of vitamin B₁₂(average diameter: 3 μm, manufactured by Rhône-Poulenc) and a lacticacid-glycolic acid copolymer (average molecular weight: 10,000; lacticacid: glycolic acid=1:1; PLGA 5010, manufactured by Wako Pure ChemicalIndustries, Ltd.) (0.9 g) is added methylene chloride (2 g), and themixture is mixed well with a bath type sonicator (Sonorex Super RK514BH,manufactured by Banderine) to give a homogeneous dispersion, which isused as an oil phase.

[0198] The oil phase is entered into a cylindrical closed vessel (innerdiameter: 110 mm, inner volume: 1 L) as shown in FIG. 4, wherein a 0.5%aqueous solution of polyvinyl alcohol (Gosenol EG-40, manufactured byThe Nippon Synthetic Chemical Industry Co., Ltd.) (400 mL) is previouslyfilled, and it is emulsified with a homogenizer (POLYTRON Homomixer;Kinematica AG, Littau, the diameter of tip: 20 mm) at 8,000 rpm for 3minutes to give an O/W type emulsion.

[0199] (2) The emulsion is stirred with a magnet stirrer (IS-3DS,manufactured by Ikeda Rika) and a Tefron-coated stirring piece (40 mm insize) at 600 rpm at room temperature, and simultaneously, methylenechloride is evaporated off from the vessel by using a cylindricalsilicone rubber hollow fiber membrane module inserted within the vessel,wherein nitrogen gas is passed through the inside of the hollow fibers.The nitrogen gas is passed at a rate of 2.4 L/minute. This treatment iscarried out for one hour.

[0200] The used cylindrical silicone rubber hollow fiber membrane moduleis the same one as used in Example 6.

[0201] (3) The resultant is treated in the same manner as described inExample 7-(3) to give red color microsphere particles (average particlesize: 29.0 μm).

[0202] By calculating in the same manner as described in Example 1-(3)and Example 5-(3), the content of vitamin B₁₂ and the content ofmethylene chloride in the microsphere particles were 7.3% and 12,900ppm, respectively, and 73.0% of vitamin B₁₂ used was taken into themicrosphere particles.

Example 9

[0203] (1) To a mixture of fine particles (0.1 g) of vitamin B₁₂(average diameter: 3 μm, manufactured by Rhône-Poulenc) and a lacticacid-glycolic acid copolymer (average molecular weight: 10,000; lacticacid: glycolic acid=1:1; PLGA 5010, manufactured by Wako Pure ChemicalIndustries, Ltd.) (0.9 g) is added methylene chloride (2 g), and themixture is mixed well with a bath type sonicator (Sonorex Super RK514BH,manufactured by Banderine) to give a homogeneous dispersion, which isused as an oil phase.

[0204] The oil phase is added to a 0.5% aqueous solution of polyvinylalcohol (Poval 220C, manufactured by Kuraray Co., Ltd.) (4 mL), and itis emulsified with a homogenizer (POLYTRON Homomixer; Kinematica AG,Littau, the diameter of tip: 10 mm) at 2,500 rpm for 5 minutes to givean O/W type emulsion.

[0205] (2) The emulsion is poured into a cylindrical closed vessel(inner diameter: 110 mm, inner volume: 1 L) as shown in FIG. 5 wherein500 mL of water is previously added, and the mixture is stirred withfour stirring blades (diameter: 50 mm, propeller R type, manufactured byHEIDON) which are provided onto a three-one motor (BL-600, manufacturedby HEIDON) at 400 rpm at room temperature, and simultaneously, methylenechloride is evaporated off from the vessel by using a cylindricalsilicone rubber hollow fiber membrane module inserted within the vessel,wherein nitrogen gas is passed through the inside of the hollow fibersfor one hour. The nitrogen gas is passed at a rate of 3.6 L/minute.Subsequently, with continuing the passing of nitrogen gas under the sameconditions, water having a temperature of 40° C. is circulated into thejacket outside of the closed vessel with an apparatus for circulatingwater having a constant temperature (RM-6, manufactured by Rauda) toheat the emulsion for 3 hours. Moreover, with continuing the passing ofnitrogen gas under the same conditions, the temperature of thecirculating water is raised to 60° C. and the procedure for removal ofmethylene chloride is continued for one hour. After completion of theprocedure, with continuing the passing of nitrogen gas under the sameconditions, the temperature of the circulating water is lowered to about5° C., and the emulsion is cooled until about 30° C. or lower.

[0206] The used cylindrical silicone rubber hollow fiber membrane moduleis the same as used in Example 6.

[0207] (3) The resultant is treated in the same manner as described inExample 1-(3) to give red color microsphere particles (average particlesize: 57.8 μm).

[0208] By calculating in the same manner as described in Example 1-(3)and Example 5-(3), the content of vitamin B₁₂ and the content ofmethylene chloride in the microsphere particles were 4.3% and less than200 ppm, respectively, and 42.9% of vitamin B₁₂ used was taken into themicrosphere particles.

Example 10

[0209] (1) A lactic acid-glycolic acid copolymer (average molecularweight: 8,000; lactic acid: glycolic acid=1:1; RG502H; manufactured byBoehringer Ingelheim) (0.45 g) and taltirelin hydrate (TRH derivative)(50 mg) are weighed and put into a glass-made test tube, and thereto isadded a mixture of methylene chloride (specific grade reagent,manufactured by Katayama Chemical Industries, Ltd.) (2 mL) and ethanol(specific grade reagent, manufactured by Katayama Chemical Industries,Ltd.) (0.5 mL), and the mixture is mixed well with a mixer (Touch MixerMT-51, manufactured by Yamato) to give a homogeneous solution.

[0210] This solution is evaporated to dryness by using a block heater(Dry Block Bath MG-2, manufactured by Tokyo Rikakikai Co., Ltd.) heatedat about 60° C. for about 30 minutes under nitrogen current. Then, theorganic solvent therein is further evaporated off by using a scaled-downlyophilizer (Speed Back Concentrator, manufactured by SABATO) to give asolid solution.

[0211] (2) To the resulting solid solution is added methylene chloride(1 g), and the mixture is mixed well by using a mixer (Touch MixerMT-51, manufactured by Yamato) to give a homogeneous solution, which isused as an oil phase.

[0212] (3) The oil phase is entered into a cylindrical closed vessel(inner diameter: 100 mm, inner volume: 1,000 mL) as shown in FIG. 5,wherein a 0.5% aqueous solution of polyvinyl alcohol (Gosenol EG-40,manufactured by The Nippon Synthetic Chemical Industry Co., Ltd.) (400mL) is previously filled and to which a plate-type silicone rubberhollow fiber membrane module is provided, and it is emulsified with ahomogenizer (POLYTRON Homomixer; Kinematica AG, Littau, the diameter oftip: 20 mm) at 20,000 rpm for 3 minutes to give an O/W type emulsion.The plate-type silicone rubber hollow fiber membrane module used isNAGASEP M60-290L-650 of the following specifications, and during theemulsification procedure, the nitrogen gas is passed through the insideof the hollow fiber membrane at a rate of 1.2 L/minute. Effective lengthof the hollow fiber membrane: 290 mm Thickness of the hollow fibermembrane: 60 μm Inner diameter of the hollow fiber membrane: 200 μmOuter diameter of the hollow fiber membrane: 320 μm Number of hollowfibers: 650 Effective area of the hollow fiber membranes: 0.15 m²

[0213] (4) After the emulsification, the tip for emulsification isquickly taken out while the passing of the nitrogen gas is continued,and it is stirred with four stirring blades (diameter: 50 mm, propellerR type, manufactured by HEIDON) which are provided onto a three-onemotor (BL-600, manufactured by HEIDON) at 400 rpm at room temperaturefor one hour, by which the methylene chloride is removed from thevessel.

[0214] (5) After completion of the above procedure, the contents of theclosed vessel (dispersion of microspheres) are passed through astainless mesh filter (opening size: 20 μm), and transferred into a 50mL volume Teflon-made centrifuge tube, which is centrifuged with acentrifugal separator (KN-30F, manufactured by Kubota Shoji K. K.) at2,000 rpm for 10 minutes, and the supernatant is discharged. Theprecipitated particles are washed with a small amount of water, andsubjected to the centrifugation like the above. This centrifugation isrepeated three times, and the finally obtained microsphere particles aretransferred to a glass-made sample tube (the full volume: 5 mL,manufactured by Iuchi Seieido) and thereto is added a small amount ofdistilled water, and the mixture is lyophilized with a lyophilizer(RLE-52ES, manufactured by Kyowa Shinku Co.) at 20° C. (outer pressure:not more than 0.3 kPa) for 15 hours to give white color microsphereparticles (average particle size: 7 μm).

[0215] (6) The microsphere particles thus obtained (0.01 g) were weighedand dissolved in acetonitrile (3 mL), and thereto was added a 0.035 Mformate buffer (pH 3.0, 2 mL), and the mixture was centrifuged at 2,000rpm for 5 minutes. The supernatant (200 μL) was measured by HPCL method,and based on a calibration curve previously prepared, the content of themedicament is estimated, and then in the light of the weight ofmicrosphere particles used, the content of the medicament in themicrosphere particles is calculated. As a result, it was 8.9%. Besides,when it was calculated based on the amount of the medicament used, thecontent of the medicament in the microsphere particles and the producedamount of microsphere particles, 89% of the medicament used was takeninto the microsphere particles.

[0216] (7) The microsphere particles thus obtained (0.02 g) weredissolved in chloroform (for high performance liquid chromatography,manufactured by Kanto Kagaku K. K.) (1 mL) to give a test sample liquid.The test sample liquid (2 μL) was measured with a gas chromatogramapparatus (the main body GC-14B, Integrator CR-7A, manufactured byShimadzu Corporation) [column packing; Gaschropack 54 (manufactured byGL Science), column temperature: 150° C.; the detector: FID; injectiontemperature: 170° C.; mobile gas: nitrogen gas; flow rate: 60 mL/h], andbased on a calibration curve previously prepared with a standardsolution of methylene chloride in chloroform, the concentration of thetest sample liquid was estimated, and then in the light of the weight ofmicrosphere particles used, the content of the methylene chloride in themicrosphere particles was calculated. As a result, it was belowdetection limits (i.e., below 100 ppm).

Example 11

[0217] To a solid solution prepared in the same manner as described inExample 10-(1) (double amount of Example 10-(1)) is added methylenechloride (2 g), and the mixture is mixed well by using a mixer (TouchMixer MT-51, manufactured by Yamato) to give a homogeneous solution,which is used as an oil phase. The oil phase is treated in the samemanner as described in Example 10-(3) to -(5) except that the amount ofa 0.5% aqueous solution of polyvinyl alcohol is the same as that inExample 10-(3) to give white color microsphere particles (averageparticle size: 7 μm).

[0218] By calculating in the same manner as described in Example 10-(6)and -(7), the content of the medicament and the content of methylenechloride in the microsphere particles were 10% and 130 ppm,respectively, and 100% of the medicament used was taken into themicrosphere particles.

Example 12

[0219] To a solid solution prepared in the same manner as described inExample 10-(1) (4-fold amount of Example 10-(1)) is added methylenechloride (4 g), and the mixture is mixed well by using a mixer (TouchMixer MT-51, manufactured by Yamato) to give a homogeneous solution,which is used as an oil phase. The oil phase is treated in the samemanner as described in Example 10-(3) to -(5) except that the amount ofa 0.5% aqueous solution of polyvinyl alcohol is the same as that inExample 10-(3) to give white color microsphere particles (averageparticle size: 7 μm).

[0220] By calculating in the same manner as described in Example 10-(6)and -(7), the content of the medicament and the content of methylenechloride in the microsphere particles were 10% and 270 ppm,respectively, and 100% of the medicament used was taken into themicrosphere particles.

Example 13

[0221] A polylactic acid (average molecular weight: 20,000; R202H;manufactured by Boehringer Ingelheim) (0.225 g) and leuprorelin acetate(LHRH derivative, manufactured by BACHEM AG) (25 mg) are weighed and putinto a glass-made test tube, and thereto is added a mixture of methylenechloride (specific grade reagent, manufactured by Katayama ChemicalIndustries, Ltd.) (1.3 mL) and ethanol (specific grade reagent,manufactured by Katayama Chemical Industries, Ltd.) (0.25 mL), and themixture is mixed well with a mixer (Touch Mixer MT-51, manufactured byYamato) to give a homogeneous solution.

[0222] This solution is evaporated to dryness by using a block heater(Dry Block Bath MG-2, manufactured by Tokyo Rikakikai Co., Ltd.) heatedat about 60° C. for about 30 minutes under nitrogen current. Then, theorganic solvent therein is evaporated off by using a scaled-downlyophilizer (Speed Back Concentrator, manufactured by SABATO) to give asolid solution.

[0223] The resulting solid solution is treated in the same manner asdescribed in Example 10-(2) to -(5) except that the amounts of methylenechloride and a 0.5% aqueous solution of polyvinyl alcohol are the sameas those in Example 10. After completion of the above procedure, thecontents of the closed vessel (dispersion of microspheres) are passedthrough a polyvinylidene difluoride membrane filter (membrane diameter:25 mm, membrane pore size: 5 μm, Durapore SVLP025, manufactured by NihonMillipore Corporation) to separate microsphere particles. The resultingmicrosphere particles are transferred to a glass-made sample tube (thefull volume: 5 mL, manufactured by Iuchi Seieido) and thereto is added asmall amount of distilled water, and the mixture is lyophilized with alyophilizer (RLE-52ES, manufactured by Kyowa Shinku Co.) at 20° C.(outer pressure: not more than 0.3 kPa) for 15 hours to give white colormicrosphere particles (average particle size: 12.9 μm).

[0224] The microsphere particles thus obtained (5 mg) are weighed andthereto is added acetonitrile (1.5 mL) and the microsphere particles aredissolved. To the solution is added a 0.5 M aqueous sodium chloridesolution (3.5 mL), and the mixture is subjected to centrifugation at2,000 rpm for 10 minutes to separate the precipitates.

[0225] The resulting supernatant (200 μL) was measured by HPLC method,and based on a calibration curve previously prepared, the content ofleuprorelin acetate (LHRH derivative) was estimated, and then in thelight of the weight of microsphere particles, the content of leuprorelinacetate in the microsphere particles was calculated. As a result, it was7.5%.

[0226] Besides, when it was calculated based on the amount ofleuprorelin acetate used, the content of leuprorelin acetate in themicrosphere particles and the produced amount of microsphere particles,84.5% of leuprorelin acetate used was taken into the microsphereparticles.

[0227] Besides, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example10-(7). As a result, it was 520 ppm.

Comparative Example 1

[0228] (1) The emulsion obtained in Example 1-(1) is poured into aclosed vessel (inner diameter: 80 mm, inner volume: 800 mL) as shown inFIG. 1 wherein 500 mL of water is previously added, and it is stirredwith four stirring blades (diameter: 50 mm, propeller R type,manufactured by HEIDON) which are provided onto a three-one motor(BL-600, manufactured by HEIDON) at 400 rpm at room temperature for 3hours.

[0229] (2) The contents of the closed vessel are treated in the samemanner as described in Example 1-(3) to give microsphere particleshaving an average particle size of 80 μm.

[0230] Besides, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example1-(3). As a result, it was 31,000 ppm.

Comparative Example 2

[0231] (1) The emulsion obtained in Example 1-(1) is poured into aclosed vessel (inner diameter: 80 mm, inner volume: 800 mL) as shown inFig. 1 wherein 500 mL of water is previously added, and it is stirredwith four stirring blades (diameter: 50 mm, propeller R type,manufactured by HEIDON) which are provided onto a three-one motor(BL-600, manufactured by HEIDON) at 400 rpm at room temperature for 2hours.

[0232] (2) The contents of the closed vessel are treated in the samemanner as described in Example 1-(3) to give microsphere particleshaving an average particle size of 73 μm.

[0233] Besides, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example1-(3). As a result, it was 39,000 ppm.

Comparative Example 3

[0234] (1) The emulsion obtained in Example 1-(1) is poured into aclosed vessel (inner diameter: 80 mm, inner volume: 800 mL) as shown inFIG. 1 wherein 500 mL of water is previously added, and it is stirredwith four stirring blades (diameter: 50 mm, propeller R type,manufactured by HEIDON) which are provided onto a three-one motor(BL-600, manufactured by HEIDON) at 400 rpm at room temperature for onehour.

[0235] (2) The contents of the closed vessel are treated in the samemanner as described in Example 1-(3) to give microsphere particleshaving an average particle size of 77 μm.

[0236] Besides, the content of methylene chloride in the microsphereparticles was calculated in the same manner as described in Example1-(3). As a result, it was 50,000 ppm.

Comparative Example 4

[0237] (1) The emulsion obtained in Example 6-(1) is poured into aclosed vessel (inner diameter: 110 mm, inner volume: 1 L) as shown inFIG. 2 wherein 500 mL of water is previously added, and it is stirredwith a magnet stirrer (IS-3DS, manufactured by Ikeda Rika) and aTeflon-made stirring piece (40 mm in size) at 600 rpm at roomtemperature for one hour.

[0238] (2) The contents of the closed vessel were treated in the samemanner as described in Example 1-(3), but microsphere particles couldnot be obtained since the content was not solidified.

Comparative Example 5

[0239] (1) The emulsion obtained in Example 7-(1) is stirred with fourstirring blades (diameter: 50 mm, propeller R type, manufactured byHEIDON) which are provided onto a three-one motor (BL-600, manufacturedby HEIDON) in a closed vessel (inner diameter: 80 mm, inner volume: 800mL) as shown in FIG. 3 at 400 rpm at room temperature for 1.5 hour.

[0240] (2) The contents of the closed vessel are treated in the samemanner as described in Example 7-(3) to give microsphere particleshaving an average particle size of 35.4 μm.

[0241] Besides, by calculating in the same manner as described inExample 1-(3) and Example 5-(3), the content of vitamin B₁₂ and thecontent of methylene chloride in the microsphere particles were 7.9% and22,600 ppm, respectively, and 79.2% of vitamin B₁₂ used was taken intothe microsphere particles.

[0242] Experiment 1

[0243] Into a closed vessel (inner diameter: 110 mm, inner volume: 1 L)as shown in FIG. 2, a 1% aqueous methylene chloride solution (1 L) wasadded, and it was stirred with a magnet stirrer (IS-3DS, manufactured byIkeda Rika) and a Teflon-made stirring piece (40 mm in size) at 600 rpmat room temperature, and simultaneously, methylene chloride wasevaporated off from the vessel by using a cylindrical silicone rubberhollow fiber membrane module (NAGASEP M60-1800, cylindrical shape)inserted within the vessel, wherein nitrogen gas was passed through theinside of the hollow fiber membrane. The nitrogen gas was passed at arate of 130 mL, 520 mL, 1.8 L, and 4 L/minute.

[0244] With the lapse of constant time, a small amount of the testsample liquid was taken at each time, and the concentration of methylenechloride in the sample liquid was measured with a gas chromatogramapparatus (measured in the same manner as described in Example 1,provided that the concentration of methylene chloride in the sampleliquid was calculated with a calibration curve prepared with an aqueousmethylene chloride solution). Based on the change of the concentrationthereof, the percentage of the concentration of methylene chloride atthe time of sampling to the concentration of methylene chloride beforethe passing of a gas (1% of the aqueous solution being calculated as100), and the data in correlation with the time were plotted. The graphthus obtained is shown in FIG. 6.

[0245] Experiment 2

[0246] The taltirelin-containing microsphere preparations obtained inExample 10, 11 and 12 were considered as Microsphere 1, 2, 3,respectively, and 10 mg of each Microsphere was precisely weighed andtransferred to a test tube with a cap (volume: 15 mL). To the tube wasadded a 1/30 M phosphate buffer (pH 7.4, 10 mL), and sealed. The tubewas put onto a rotating cultivator (RT-50, manufactured by TAITEC) in anair thermostatic chamber (Biochamber BC-1200, manufactured by TAITEC)controlled at 37° C., and shaken at a rotational speed of 25 rpm. Withthe lapse of prescribed time, the eluate was taken, and then, additional1/30 M phosphate buffer of the same volume of the eluate taken wasadded, and further the same procedures were repeated. The amount oftaltirelin dissolving out from microspheres were calculated by HPLCmethod. The accumulated amount of taltirelin (TRH derivative) dissolvingout from microspheres at 37° C. was plotted in correlation with thedissolution period. The graph thus obtained is shown in FIG. 7.

[0247] Experiment 3

[0248] The leuprorelin acetate-containing microsphere preparationprepared in Example 13 was considered as Microsphere 4, and 5 mg thereofwas precisely weighed and transferred to test tubes with a cover(volume: 15 mL). To five test tubes with a cover thus prepared was addeda 1/30 M phosphate buffer (pH 7.0, 10 mL) containing 0.05% Tween 80(polyoxyethylene sorbitan fatty acid ester, manufactured by NikkoChemicals Co., Ltd.). Each test tube was sealed, and put onto a rotatingcultivator (RT-50, manufactured by TAITEC) in an air thermostaticchamber (Biochamber BC-1200, manufactured by TAITEC) controlled at 37°C., and shaken at a rotational speed of 25 rpm. With the lapse ofprescribed time, each one tube was taken out, and centrifuged with acentrifugal separator (KN-30F, manufactured by Kubota Shoji K. K.) at2,000 rpm for 10 minutes in order to precipitate microspheres. Thesupernatant was discharged, and in order to distill moisture off, thetube was kept in a glass-made desiccator containing silica gel underreduced pressure overnight. To the tube was added acetonitrile (1.5 mL)and the dried microsphere particles were dissolved. Then, a 0.5 Maqueous sodium chloride solution (3.5 mL) was added to the tube, and thetube was centrifuged at 2,000 rpm for 10 minutes to remove theprecipitates. The resulting supernatant (200 μL) was measured by HPCLmethod, and based on a calibration curve previously prepared, thecontent of leuprorelin acetate remained in the microspheres wasestimated. The amount of leuprorelin acetate remained in themicrospheres at 37° C. was plotted in correlation with the dissolutionperiod. The graph thus obtained is shown in FIG. 8.

INDUSTRIAL AVAILABLILITY

[0249] According to the method of the present invention, whenmicrospheres are prepared from an emulsion by an in-water drying methodwith a gas separation membrane, by supplying an emulsion from one sideof the membrane and the organic solvent is evaporated off into the otherside thereof, the organic solvent can be removed extremely efficiently,and further the procedures can be done in a closed system, and hence,the improved process of the present invention is extremely excellentalso from the environmental viewpoint.

1. A method for preparation of microsphere from an emulsion wherein anorganic phase containing an organic solvent having a boiling point lowerthan that of water and a hardly-water-soluble polymer is emulsified inan aqueous phase by an in-water drying method, which comprises: (1)using an apparatus equipped with a gas separation membrane; (2)supplying the emulsion to be subjected to in-water drying to one side ofsaid gas separation membrane; (3) evaporating off the organic solventcontained in said emulsion to the other side of said gas separationmembrane.
 2. The method for preparation of microsphere according toclaim 1, wherein a medicament is contained in the organic phase.
 3. Themethod for preparation of microsphere according to claim 2, wherein themedicament is contained in a ratio of 0.01 to 60% by weight based on theweight of the hardly-water-soluble polymer.
 4. The method forpreparation of microsphere according to claim 3, wherein in themedicament-containing organic phase, the medicament is directlydissolved or dispersed in a solution of the hardly-water-solublepolymer, or an aqueous solution of the medicament is dispersed in asolution of the hardly-water-soluble polymer, or the medicament isdissolved or dispersed in a dispersed solution of onehardly-water-soluble polymer, which solution is dispersed in a solutionof another hardly-water-soluble polymer.
 5. The method for preparationof microsphere according to any one of claims 1 to 4, wherein thehardly-water-soluble polymer is a hardly-water-soluble biodegradablepolymer.
 6. The method for preparation of microsphere according to claim5, wherein the hardly-water-soluble biodegradable polymer is a polyesterof a hydroxyfatty acid.
 7. The method for preparation of microsphereaccording to claim 6, wherein the polyester of a hydroxyfatty acid isone or more members selected from a polylactic acid, a copolymer oflactic acid-glycolic acid, and a copolymer of 2-hydroxybutyricacid-glycolic acid.
 8. The method for preparation of microsphereaccording to any one of claims 1 to 7, wherein the hardly-water-solublepolymer is contained in the organic phase in a concentration of 0.01 to90% by weight.
 9. The method for preparation of microsphere according toany one of claims 1 to 8, wherein the organic solvent having a boilingpoint lower than that of water is one or more members selected from ahalogenated aliphatic hydrocarbon solvent, an aliphatic ester solvent,an aromatic hydrocarbon solvent, an aliphatic hydrocarbon solvent, aketone solvent, and an ether solvent.
 10. The method for preparation ofmicrosphere according to claim 9, wherein the organic solvent has aboiling point lower by 15 to 60° C. than that of water under theevaporation conditions therefor.
 11. The method for preparation ofmicrosphere according to claim 9, wherein the organic solvent is onemember selected from methylene chloride, chloroform and ethyl acetate.12. The method for preparation of microsphere according to any one ofclaims 1 to 11, wherein the aqueous phase contains one or more membersselected from an emulsifying agent, a polyethylene castor oilderivative, a polyvinylpyrrolidone, a polyvinyl alcohol, acarboxymethylcellulose, a methylcellulose, a lecithin, and a gelatin.13. The method for preparation of microsphere according to any one ofclaims 1 to 12, wherein in the emulsion, the aqueous phase is containedin a volume of 1 to 10,000 times of the volume of the organic phase. 14.The method for preparation of microsphere according to any one of claims1 to 13, wherein the gas separation membrane is a pervaporation membraneor a porous membrane.
 15. The method for preparation of microsphereaccording to claim 14, wherein the gas separation membrane is apervaporation membrane.
 16. The method for preparation of microsphereaccording to claim 15, wherein the gas separation membrane is asilicon-rubber pervaporation membrane.
 17. The method for preparation ofmicrosphere according to any one of claims 14 to 16, wherein the gasseparation membrane is used in the form of a bundle of plural gasseparation membranes which form hollow fibers.
 18. The method forpreparation of microsphere according to any one of claims 1 to 17,wherein the evaporation of the organic solvent to the other side of thegas separation membrane is carried out by one means or a combination ofmeans selected from passing a gas on said other side of the gasseparation membrane, decompressing said other side of the gas separationmembrane, or warming the emulsion which is supplied to one side of thegas separation membrane.
 19. The method for preparation of microsphereaccording to claim 18, which is carried out by circulatively repeatingthe steps: taking out a portion of the emulsion; supplying the same toone side of the gas separation membrane; and then returning a resultingliquid after the evaporation of the organic solvent into the originalemulsion.
 20. The method for preparation of microsphere according toclaim 19, wherein only a portion of the aqueous phase which is obtainedby filtration of the emulsion is taken out, and then supplying the sameto one side of the gas separation membrane.
 21. The method forpreparation of microsphere according to claim 19 or 20, wherein a bundleof plural gas separation membranes which form hollow fibers is used, anda portion of the emulsion for in-water drying is introduced into theinner side of said gas separation membranes which form hollow fibers andthe organic solvent is evaporated to the outside of said gas separationmembranes.
 22. The method for preparation of microsphere according toclaim 18, wherein the bundle of gas separation membranes which formhollow fibers is immersed in the emulsion, and then passing a gas on theinner side of said gas separation membranes which form hollow fibers toevaporate the organic solvent.
 23. The method for preparation ofmicrosphere according to any one of claims 1 to 22, wherein the organicsolvent to be evaporated is recovered by cooling or by absorbing to aporous substance.
 24. The method for preparation of microsphereaccording to any one of claims 1 to 23, wherein the preparation ofmicrosphere by an in-water drying method is carried out in a closedsystem.
 25. An apparatus for preparation of microsphere by an in-waterdrying method from an emulsion wherein an organic phase containing anorganic solvent having a boiling point lower than that of water and ahardly-water-soluble polymer is emulsified in an aqueous phase, whichconsists of the following elements: (a) a vessel for filling anemulsion; (b) a gas separation membrane module for evaporating off anorganic solvent from the emulsion; (c) a circulation pathway whichconnects the vessel and the gas separation membrane module; and (d) apump for circulating the emulsion through the gas separation membranemodule.
 26. An apparatus for preparation of microsphere by an in-waterdrying method from an emulsion wherein an organic phase containing anorganic solvent having a boiling point lower than that of water and ahardly-water-soluble polymer is emulsified in an aqueous phase, whichconsists of the following elements: (a) a vessel for filling anemulsion; and (b) a gas separation membrane module to be immersed in theemulsion in the vessel for evaporating an organic solvent from theemulsion.